1.1 Technical Field
The present invention relates to methods of design or identification of biologically active compounds which methods are based on the first definition ever of a three-dimensional structure of a protein involved in the leukotriene cascade. Further, the invention relates to novel compounds obtained by said methods, to advantageous uses of such compounds as well as to processes for the preparation thereof.
1.2 Prior art
Leukotriene A4 (LTA4) hydrolase is a pivotal enzyme in the biosynthesis of leukotrienes, a family of paracrine hormones implicated in the pathophysiology of inflammatory and allergic disorders, in particular bronchial asthma (Samuelsson, B. Science 220, 568-75 (1983); and Lewis, R. A., Austen, K. F. & Soberman, R. J. N Engl J Med 323, 645-55 (1990)). Leukotrienes are formed by immunocompetent cells including neutrophils, eosinophils, basophils, mast cells, and macrophages, in response to a variety of immunological as well as non-immunological stimuli. These lipid mediators are divided into two major classes exemplified by the chemotaxin LTB4, and the spasmogenic cysteinyl-leukotrienes (LTC4, LTD4, and LTE4). Leukotriene biosynthesis is initiated by the enzyme 5-lipoxygenase which converts arachidonic acid into the unstable epoxide LTA4, a central intermediate in the leukotriene cascade. LTA4 may in turn be hydrolyzed into LTB4 by the enzyme LTA4 hydrolase, or conjugated with GSH to form LTC4, a reaction catalyzed by a specific LTC4 synthase. During cellular activation, all key enzymes in leukotriene biosynthesis, except LTA4 hydrolase, form a biosynthetic complex assembled at the nuclear membrane, suggesting that leukotrienes may have unknown intranuclear functions related to gene regulation or cell growth (Serhan, C. N., Haeggstrom, J. Z. & Leslie, C. C. Faseb J 10, 1147-58 (1996)).
Leukotriene B4, the natural product of LTA4 hydrolase, is one of the most powerful chemotactic agents known to date and triggers leukocyte adherence and aggregation at only nM concentrations (Ford-Hutchinson, A. W., Bray, M. A., Doig, M. V., Shipley, M. E. & Smith, M. J. H. Nature 286, 264-265 (1980)). Hence, this molecule is regarded as a key mediator of inflammation, and has been implicated in a number of diseases, including arthritis, psoriasis, inflammatory bowel disease (IBD), and chronic obstructive pulmonary disease (COPD). Furthermore, the role of LTB4 in inflammation has been well corroborated by the anti-inflammatory properties of LTA4 hydrolase inhibitors, particularly in combination with a cyclooxygenase inhibitor, and specific LTB4 receptor antagonists, as well as the reduced inflammatory reactions observed in several animal models of leukotriene deficiency (Tsuji, F., Miyake, Y., Enomoto, H., Horiuchi, M., Mita, S. Eur. J. Pharmacol. 346, 81-85, (1998); Chen, X. S., Sheller, J. R., Johnson, E. N. & Funk, C. D. Nature 372, 179-182 (1994); Griffiths, R. J., et al. Proc Natl Acad Sci USA 92, 517-21 (1995); and Griffiths, R. J., et al. J Exp Med 185, 1123-9 (1997)). In addition, LTB4 modulates the immune response, e.g., by interference with specific subsets of lymphocytes, production of cytokines, as well as liberation of immunoglobulins from B-lymphocytes (Payan, D. G., Missirian-Bastian, A. & Goetzl, E. J. Proc Natl Acad Sci USA 81, 3501-5 (1984); Rola-Pleszczynski, M. & Lemaire, I. J Immunol 135, 3958-61 (1985); and Yamaoka, K. A., Claesson, H. E. & Rosen, A. J Immunol 143, 1996-2000 (1989)). Recent data also indicate that LTB4 stimulates, and thus has a crucial role in the regulation of, cell proliferation and cell survival in HL-60 cells, suggesting that LTA4 hydrolase inhibitors may have an anti-proliferative effect. (Dittman, K. H., Mayer, C., Rodemann, H. P., Petrides, P. E., and Denzlinger, C. Leuk Res. 22, 49-53 (1998)). The cell surface receptor for LTB4 (BLTR) was recently cloned and found to be abundantly expressed in the immune system, including lymphocytes, spleen and thymus (Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y. & Shimuzu, T. Nature 387, 620-624 (1997)). BLTR belongs to a family of chemokine receptors and, interestingly, together with CD4 it was found to be an efficient coreceptor for HIV-1 infection (Owman, C., et al. Proc Natl Acad Sci USA 95, 9530-4 (1998)). Moreover, LTB4 is also a natural ligand to the nuclear orphan receptor PPARα, suggesting that LTB4 may have intranuclear functions possibly related to lipid homeostasis (Devchand, P. R., et al. Nature 384, 39-43 (1996)).
LTA4 hydrolase is a cytosolic 69 kDa enzyme without any similarity to other soluble or membrane bound xenobiotic epoxide hydrolases (Funk, C. D., et al. Proc Natl Acad Sci USA 84, 6677-81 (1987)). The enzyme's epoxide hydrolase activity, which generates LTB4, is highly substrate selective accepting only LTA4 and to a small extent the double bond isomers LTA3 and LTA5. Typically, LTA4 hydrolase undergoes suicide inactivation and covalent modification when exposed to LTA4 (Evans, J. F., Nathaniel, D. J., Zamboni, R. J. & Ford-Hutchinson, A. W. J. Biol. Chem. 260, 10966-10970 (1985)). During this process, LTA4 apparently binds to Tyr-378, a residue which also seems to play a role for the formation of the critical cis-trans-trans geometry in the conjugated triene structure of LTB4 (Mueller, M. J., et al. Proc Natl Acad Sci USA 93, 5931-5935 (1996); and Mueller, M., Andberg, M., Samuelsson, B. & Haeggstrom, J. Z. J. Biol. Chem. 271, 24345-24348 (1996)).
From sequence comparisons with certain metalloproteases and aminopeptidases, a zinc binding motif (HEXXH-X18-E) was unexpectedly found in LTA4 hydrolase (Vallee, B. L. & Auld, D. S. Proc. Natl. Acad. Sci. USA 87, 220-224 (1990)). Further studies demonstrated that the enzyme indeed contains one catalytic zinc atom complexed to His295, His299, and Glu318 (Medina, J. F., et al. Proc. Natl. Acad. Sci. USA 88, 7620-7624 (1991)). In addition, a previously unknown peptide cleaving activity was discovered which requires the presence of anions, particularly chloride (Haeggström, J. Z., Wetterholm, A., Medina, J. F. & Samuelsson, B. J Lipid Mediator 6, 1-13 (1993)). Although the endogenous physiological peptidase substrate(s) has not yet been identified, LTA4 hydrolase cleaves certain arginyl di- and tripeptides with very high efficiency (Örning, L., Gierse, J. K. & Fitzpatrick, F. A. J. Biol. Chem. 269, 11269-11273 (1994)). Hence, LTA4 hydrolase can be described as a bifunctional zinc metalloenzyme with the unique ability to accept both lipid and peptide substrates. Using site-directed mutagenesis, Glu296 and Tyr383 were found to be critical for the peptidase reaction, presumably as a general base and proton donor, respectively (Blomster, M., Wetterholm, A., Mueller, M. J. & Haeggström, J. Z. Eur. J. Biochem. 231, 528-534 (1995); and Wetterholm, A., et al. Proc Natl Acad Sci USA 89, 9141-9145 (1992)). Since the enzyme's ability to convert LTA4 into LTB4 was not affected by the mutations, the two enzyme activities of LTA4 hydrolase are exerted via non-identical but overlapping active sites. Notably, unlike other enzymes in the leukotriene cascade, LTA4 hydrolase is ubiquitous in mammalian cells and tissues suggesting that it may have other functions presumably related to its peptide cleaving activity.
As a consequence of the identification of LTA4 hydrolase as a zinc metalloenzyme with a peptidase activity, it was observed that LTA4 hydrolase is inhibited by bestatin, a general aminopeptidase inhibitor, and captopril, an inhibitor of angiotensin converting enzyme (Örning, L., et al. J. Biol. Chem. 266, 16507-16511 (1991)).
Tsuge et al., (J. Mol. Biol. 238, 854-856 (1994)), have described the crystallization of LTA4 hydrolase. However, despite the well recognized need thereof, the three-dimensional structure of LTA4 hydrolase has not yet been disclosed. More specifically, the problems that need to be overcome in order to provide such a determination may in brief be explained as follows. There are two major difficulties in obtaining a three-dimensional structure of a protein molecule. The first one is to grow crystals of good quality that are reproducible and diffract to atomic resolution (beyond 2.5 Å). This means a thorough and cumbersome investigation of parameters that influence the crystal growth such as pH, temperature, nature of buffers, nature of precipitant, just to mention a few. The addition of ligands such as substrate analogues or inhibitors or the addition of other molecules can be important for obtaining good crystals. There is only little understanding of the physical background of the crystallisation process which means that the search for suitable crystallisation conditions for a certain protein is unique, requires creativity and intuition, and is governed by trial and error procedures. The purity of the protein is also a crucial parameter in the crystallisation and a suitable degree of purity can be hard, or even impossible, to achieve. The second major difficulty is associated with overcoming the phase-problem which is inherent to X-ray diffraction methods. To be able to overcome this problem it is necessary to substitute the protein with suitable heavy atom substance such as e.g. mercury, gold or platinum compounds. Crystals often cannot withstand the treatment with these compounds and the search for suitable substitutions is not straight forward and may become very exhaustive. Another option is to substitute all methionines by seleno-methionine (Se-Met) residues. This method requires production of recombinant protein in special strains of E. coli under non-standard conditions, followed by a new purification and recrystallisation of the Se-Met containing protein. Although Tsuge et al reported the crystallisation of LTA4 hydrolase, their crystals only diffracted to medium resolution and the phase-problem was not solved. Thus, as a reliable definition of the three-dimensional structure of LTA4 hydrolase would enable e.g. a display in visual form on a computer screen of the shape of the molecule, then, could the above mentioned problems be solved, a whole range of possibilities would be opened, such as rational structure-based drug design, e.g. in combination with combinatorial chemistry, aimed at production of novel medicaments useful in disorders associated with the leukotriene cascade, as well as protein-engineering to create novel variants of the enzyme with altered, but yet useful, catalytic properties.
As LTA4 hydrolase is a recognized important drug target, some inhibitors thereof have been synthesized (Wetterholm, A., et al. J Pharmacol Exp Ther 275, 31-7 (1995); and Yuan, W., Wong, C., Haeggstrom, J. Z., Wetterholm, A. & Samuelsson, B. J. Am. Chem. Soc., 114, 6552-6553 (1992)). Interestingly, certain inhibitors of LTA4 hydrolase were reported to act also as LTB4 receptor antagonists (Labaudinière R, Hilboll G, Leon-Lomeli A, Terlain B, Cavy F, Parnham M, Kuhl P, and Dereu N. J. Med. Chem. 35, 3170-3179 (1992)). Due to the absence of any available information regarding the three-dimensional structure of LTA4 hydrolase, as discussed above, none of the previously described inhibitors have been designed based on the exact structure thereof. Accordingly, there is a need within this field of determining the three-dimensional structure of LTA4 hydrolase in order to design more potent and selective inhibitors of LTA4 hydrolase as well as modified structures exhibiting even more advantageous pharmaceutical properties.